The search for new compounds in many cases makes use of large libraries of compounds to screen and identify a compound that has a desired activity or characteristic. Combinatorial peptide library technology is a valuable resource for drug discovery and development. Recombinant peptide libraries displayed on phage or other viral particles have proven especially useful in such screens. Numerous groups are working to develop biologically active peptides obtained from peptide libraries in the search of novel treatments for many human diseases and illnesses.
Phage-displayed peptide library technology has been widely used in the search of novel treatments for many human diseases and illnesses. Peptide libraries displayed on filamentous phage have been used as a screening resource for identifying peptides bound to any given target thereby showing pharmacologic effects. Peptides so identified can subsequently be synthesized in bulk using conventional synthetic chemistry methods.
The bacteriophage M13 is a non-enveloped filamentous Escherichia coli phage of the Inoviridae family with a single stranded (ss)DNA genome. The nucleocapsid consists of four bacteriophage proteins with different copy numbers: pVIII approximately 2700 copies while pIII, pVI and pIX are present in 5 copies. Among other, bacteriophages like T4, T7, fd and lambda, M13 has been successfully used for phage display for use in a biotechnological screening method. In such a screening approach a random library of peptides is presented on the surface of the nucleocapsid of the phage M13 to study interaction of the different phages with a binding partner (protein-protein, protein-DNA, etc). Usually synthetic oligonucleotides are cloned into genes coding for proteins which constitute the nucleocapsid and thereby the peptide of interest (or a library of different peptides) is presented on the surface of the phage M13 nucleocapsid for subsequent binding studies.
The phage display methods typically involve the insertion of random oligonucleotides into a phage genome such that they direct a bacterial host to express peptide libraries fused to phage coat proteins (e. g., filamentous phage pIII, pVI or pVIII). The advantages of this technique are in the small dimension of the phage allowing to handle libraries with up to 1015 different individuals and in the physical linkage of the displayed peptides with the genetic information that encode them.
The basic phage display technology has been expanded to include peptide libraries that are displayed from replicable genetic packages other than phage, such as eukaryotic viruses, bacteria and yeast cells. The principles and strategy are closely analogous to those employed for phage, namely, that nucleic acids encoding peptides to be displayed are inserted into the genome of the package to create a fusion protein between the peptides to be screened and an endogenous protein that is exposed on the cell or viral surface. Expression of the fusion protein and transport to the cell surface results in display of peptides at the cell or viral surface.
In an effort to increase diversity of a library though a secondary peptide structure some groups have produced conformationally-constrained peptides through chemical reactions.
EP1187914B1 discloses a library of structurally-constrained peptides comprising a plurality of cyclic peptides stabilized through disulfide bridges between cystein residues.
WO2009/098450A2 discloses a phage particle displaying a disulfide-stabilized bicyclic peptide linked via a connector compound.
US2009/0137424A1 discloses the posttranslational modification of phage displayed polypeptides, which contain unnatural amino acids to provide targets for azide-alkyne [3+2]cycloaddition reactions and Staudinger modifications.
The biosynthesis of depsipeptide natural compounds is described in EP2048155A1. A precursor peptide sequence of between 5 and 50 amino acids is modified by an ATP-grasp-like enzyme. The nucleic acid molecule is introduced e.g. into E. coli to produce the depsipeptide natural compounds, for example microviridin.
Natural product synthesis of several ribosomal peptides is reviewed by Oman et al (Nature Chemical Biology 6:9-18 (2010)). Precursor peptides are post-translationally processed mediated by leader peptides. These leader peptides assist in folding the precursor peptide, stabilizing the precursor against degradation and keeping the precursor inactive during the biosynthesis inside the host until the appropriate time for secretion and proteolysis.
There is a pressing need for new targeting peptides developed through peptide library technology. It is, thus, the objective of the present invention to provide an improved library of structurally constrained peptides for screening purposes.